Structure of Rift Valley Fever Virus RNA-Dependent RNA Polymerase

Abstract

Rift Valley fever virus (RVFV) belongs to the order Bunyavirales and is the type species of genus Phlebovirus , which accounts for over 50% of family Phenuiviridae species. RVFV is mosquito-borne and causes severe diseases in both humans and livestock, and consists of three segments (S, M, L) in the genome. The L segment encodes an RNA-dependent RNA polymerase (RdRp, L protein) that is responsible for facilitating the replication and transcription of the virus. It is essential for the virus and has multiple drug targets. Here, we established an expression system and purification procedures for full-length L protein, which is composed of an endonuclease domain, RdRp domain, and cap-binding domain. A cryo-EM L protein structure was reported at 3.6 Å resolution. In this first L protein structure of genus Phlebovirus , the priming loop of RVFV L protein is distinctly different from those of other L proteins and undergoes large movements related to its replication role. Structural and biochemical analyses indicate that a single template can induce initiation of RNA synthesis, which is notably enhanced by 5’ viral RNA. These findings help advance our understanding of the mechanism of RNA synthesis and provide an important basis for developing antiviral inhibitors. Importance The zoonosis RVF virus (RVFV) is one of the most serious arbovirus threats to both human and animal health. RNA-dependent RNA polymerase (RdRp) is a multifunctional enzyme catalyzing genome replication as well as viral transcription, so the RdRp is essential for studying the virus and has multiple drug targets. In our study, we report the structure of RVFV L protein at 3.6 Å resolution by cryo-EM. This is the first L protein structure of genus Phlebovirus. Strikingly, a single template can initiate RNA replication. The structure and assays provide a comprehensive and in-depth understanding of the catalytic and substrate recognition mechanism of RdRp.

Structural insights into viral genome replication by the severe fever with thrombocytopenia syndrome virus L protein

“…Recently, cryo-electron microscopy (cryo-EM) data from single-particle analysis became available describing relevant functional states of Machupo virus (MACV), LACV and LASV L proteins in complex with vRNA, emphasizing how these complex molecular machines undergo major conformational changes during viral genome replication and transcription (13,14,27,28). However, to date, structures of the SFTSV and Rift valley fever virus (RVFV) L proteins, as models for phenuiviruses, are only available in an apo state, i.e., without vRNA (17,(29)(30)(31)(32)(33). These structures demonstrated that the overall architecture of the phenuivirus L protein was similar to the structure of the heterotrimeric influenza virus polymerase complex and the peribunyavirus LACV L protein but also identified some notable differences between bunyavirus and influenza virus polymerases, the most apparent being differences in the fingertip's insertion (SFTSV L protein residues 913 -920).…”

Identification of Single Amino Acid Changes in the Rift Valley Fever Virus Polymerase Core Domain Contributing to Virus Attenuation In Vivo

“…Here we focused on those in residues 924, 1050 and 1303 because of their location in the catalytic core of the RNA-dependent RNA polymerase (RdRp). In particular, residue 924 is located within motif F in the RdRp core, a motif involved in the binding of the incoming rNTP ( te Velthuis, 2014 ; Ferron et al., 2017 ; Wang et al., 2021 ) as well as in the interaction of favipiravir with the viral polymerase. A small number of viruses has been isolated with altered sensitivity to favipiravir that share the substitution of a conserved K within the F-motif, associated to a cost to viral fitness that can be restored by accompanying mutations, and these changes affect the polymerase activity and maybe also its fidelity ( Delang et al., 2014 ; Abdelnabi et al., 2017 ; Goldhill et al., 2018 ).…”

“…Since G924 is structurally located between the two key positive-charged residues K918 and R926 conserved in F-motifs of RdRps from different RNA-virus families ( te Velthuis, 2014 ; Peng et al., 2021 ), it seemed reasonable to attribute a significant role to the G924S substitution and its combination with I1050V and/or A1303T. The recent cryo-EM model of RVFV RdRp locates residue 924 in the fingertips, residue 1050 within the finger node and 1303 within the thumb ( Wang et al., 2021 ), but no precise locations or interactions nor an obvious structural/physical connection among the three residues provide a clue about their role ( Figure 6 ).…”

“… Location of the three positions under study in the representation of RVFV L-protein by PyMOL, as defined by the cryo-EM model (accession code 7EEI, ( Wang et al., 2021 ). …”

“…Based on this high degree of sequence identity, the change of polymerase residues involved in key steps of viral replication has been proposed as a universal approach to obtain attenuated RNA viruses for vaccine purposes ( Vignuzzi et al., 2008 ; Weeks et al., 2012 ; Yeh et al., 2020 ; Mori et al., 2021 ). Recently, the structure of the RVFV L-protein has been solved by cryo-EM, providing the precise location of most of these structural motifs and residues ( Wang et al., 2021 ).…”